JP2015186488A - Multilayer tissue culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To disclose a vessel for culturing cells.SOLUTION: The vessel includes: a bottom including a base with an upwardly extending wall at least partially bounding the base of the bottom; a top including a base with a downwardly extending wall at least partially bounding the base of the top; a tubular neck with an opening defined therein; and one or more shelves, each shelf including a base with an upwardly extending wall at least partially bounding the base of the shelf. The upwardly extending wall of a first shelf adjoins the downwardly extending wall of the top, the first shelf being located between the bottom and the top. The base of each of the shelves has at least one aperture formed therein. The bottom, the top, and the one or more shelves collectively define an enclosed volume for culturing cells. The tubular neck extends from the vessel with the enclosed volume being accessible by the opening in the tubular neck. Advantageously, this vessel provides high volume cell culture in a manner that increases efficiency and reduces the cost of culturing cells.

Description

  The present invention relates to a laboratory container. More particularly, the present invention relates to multilayer tissue culture containers.

  Cells (cells) such as eukaryotic cells are cultured for a variety of purposes, including basic research and high throughput screening. However, culturing cells under sterile conditions is cumbersome and expensive. Thus, there is a need for a more efficient and cost effective laboratory vessel for culturing cells.

  The present invention discloses a container for culturing cells. The container includes a bottom including a base, the bottom having an upwardly extending wall that at least partially defines a boundary of the bottom base, and a top including the base, the boundary of the top base being at least partially A top having a downwardly extending wall, a tubular neck defining an opening, and one or more shelves, each shelf above the base and at least partially defining the boundary of the base of the shelf And an extended wall. A wall extending above the first shelf is adjacent to a wall extending below the top, and the first shelf is located between the bottom and the top. The base of each shelf has at least one hole formed in it. The bottom, top, and one or more shelves aggregate to define an enclosed volume for culturing cells. A tubular neck extends from the container and the enclosed volume is accessible by an opening in the tubular neck. Advantageously, the container provides a large amount of cell culture in a manner that increases efficiency and reduces the cost of cell culture.

  The container of the present invention increases the total culture area per surface area of the bottom area (footprint) of the culture container. The vessel also increases the percent recovery of cells per surface area of the bottom area of the culture vessel. Thus, the container provides a means for efficient mass cell culture. Such containers are used in a manual and / or automated manner. Exemplary embodiments include, but are not limited to, a container that maintains the total bottom area of a standard BD Falcon T-175 flask, and thus the Automation Partnership's SelectT ™ and CompacT ™ automated cell culture systems. Etc., and compatible with an automated cell culture system. The container, however, can be enlarged or reduced compared to the height of a standard flask such as a BD Falcon T-175 flask. In particular, the stacked arrangement of shelves in the container allows the height to be changed so that the surface area for culturing cells is expanded.

  In addition, the container design minimizes the number of operations required to fill and remove the media, thereby increasing the efficiency of culturing cells and reducing the chance of container contamination with each transfer. Preferably, the container includes an opening that is large enough for the pipette to access the rear wall of the container, thereby facilitating good laboratory activity for good cell culture techniques. Exemplary embodiments include, but are not limited to, a container with an opening that is large enough for a 10 mL pipette or even a 50 mL pipette to access the back wall.

  The container design also reduces the amount of media and cells “hung up” along the walls and corners in the container, allowing efficient removal of the media and cells. The container design also efficiently distributes liquid (eg, medium, phosphate buffered saline (PBS), trypsin) to different cell layers, thereby reducing the amount of liquid required and Facilitates further distribution and facilitates further distribution of cells during seeding. This provides a more consistent nutrient distribution, nutrient consumption rate, cell growth rate, and dissociation rate at the time of cell harvest, which results in more constant cell growth and / or differentiation and even healthier whole cells A healthy population. Such containers also provide significant cost savings with respect to the amount of media and dissociator (eg, trypsin) required for cell growth and cell dissociation, respectively. In short, the container of the present invention reduces the amount of labor and expense associated with culturing cells.

  These and other features of the present invention will be better understood through a review of the following detailed description and accompanying drawings.

1 is a perspective view of a container formed in accordance with the present invention. FIG. 2 is a cross-sectional view taken along line 2-2 in FIG. It is a top view of a container. FIG. 4 is a cross-sectional view taken along line 4-4 of FIG. It is a side view of a container. It is a perspective view of the bottom part which can be used by this invention. FIG. 7 is a cross-sectional view taken along line 7-7 in FIG. It is a perspective view of the top part which can be used by this invention. FIG. 9 is a cross-sectional view taken along line 9-9 in FIG. It is sectional drawing along the 10-10 line of FIG. It is a perspective view of the shelf part which can be used by this invention. FIG. 12 is a cross-sectional view taken along line 12-12 of FIG. 1 is a perspective view of a container formed in accordance with the present invention. FIG. 14 is a sectional view taken along line 14-14 in FIG. 13. It is a perspective view of the shelf part which can be used by this invention. FIG. 2 is a schematic diagram showing the equilibrium of a cell medium in a container formed in accordance with the present invention. FIG. 3 is a schematic diagram illustrating pipette access in a container formed in accordance with the present invention. FIG. 2 is a schematic diagram showing a layered arrangement of cell media in a container formed in accordance with the present invention. It is a top view of the shelf which can be used by this invention. FIG. 20 is a cross-sectional view taken along line 20-20 in FIG. It is an enlarged view of the division 21 of FIG. It is an enlarged view of the division | segmentation 22 of FIG. It is a figure which shows a different storage wall shape. It is a figure which shows a different storage wall shape. It is a figure which shows the cap which can be used by this invention. It is a figure which shows the cap which can be used by this invention. It is a figure which shows the cap which can be used by this invention.

  Referring to the figure, a container 10 for cell culture is depicted. As will be appreciated by those skilled in the art, the container 10 can be utilized in a variety of cell cultures. Container 10 generally includes a bottom 12, a top 14, a tubular neck 16, and one or more shelves 20. Tubular neck 16 has an opening 18 defined therein. One or more shelves 20 are disposed between the bottom 12 and the top 14. The bottom 12, top 14 and shelf 20 collectively define an enclosed volume 22 for culturing cells. The tubular neck 16 extends from the container 10 and the enclosed volume 22 is accessible by an opening 18 in the tubular neck 16.

  The bottom 12 is generally shaped like a tray and has a base 24. A wall 26 extends upward from the base 24. Wall 26 at least partially bounds base 24. Preferably, the wall 26 completely borders the base 24 at the periphery.

  Depending on the desired overall size of the container, one or more shelves 20 can be utilized. When there is much quantity of the shelf part 20, the cell culture capacity of the container 10 will increase. Excessive size is not desired due to limitations in gas flow and distribution. Embodiments utilizing two or four shelves 20 are conceivable. However, other quantities can be used. Each shelf 20 is generally shaped like a basin, has a base 28, has a wall 30 extending upwardly from the base 28, and the wall 30 at least partially borders the base 28. Preferably, for each shelf 20, the wall 30 completely borders the base 28 at the periphery. In addition, at least one hole 32 is formed through the base 28 of each shelf 20. Preferably, each shelf 20 includes at least two holes 32, a gas transmission hole 32a and a flow hole 32b. As will be discussed further below, the gas flow holes 32a allow gas transmission through the shelf 20 so that the gas flow can be distributed throughout the container 10. The flow holes 32b are cells disposed within the container 10 to distribute cell media between the various support layers of the container 10 as defined by the bottom 12 and the shelf 20. Provided to balance the medium. Although it is preferred to utilize a two-hole configuration for each shelf 20, a single hole 32 is provided in each shelf 20 that can perform both gas transfer and balance functions. May be. Furthermore, more than two holes 32 may be utilized for one or both functions of gas transfer and balance. It is preferable that the gas transmission hole 32a and the flow hole 32b of each shelf part 20 are separated.

  The top 14 has a generally inverted basin shape and has a base 34 and a wall 36 that extends downwardly from the base 34 and at least partially bounds the base 34. Preferably, a tubular neck 16 is formed integrally with the top 14 and extends from the top 14. Alternatively, an opening may be formed in the top 14 and the tubular neck 16 may be formed as a separate part and secured to the top 14 using any known technique such as welding or gluing. It is further possible to form an opening extending from the top 14 into one or more shelves 20 to which the tubular neck 16 is attached. The opening 18 of the tubular neck 16 may be elliptical (including circular) or semi-circular and can be formed in a constant or variable shape along the length of the tubular neck 16 (eg, tubular). Along the length of the neck 16, part of the opening 18 is elliptical and part is semicircular).

  The bottom 12, top 14, and one or more shelves 20 are arranged in a stacked manner so as to assemble to form a container 10. Preferably, the component is formed from a thermoplastic material that is compatible with the cells of interest. Polystyrene may be utilized. Other materials may be utilized as will be appreciated by those skilled in the art. The component can be made transparent, lightly colored (eg, blue), or colored (eg, amber). In addition, various portions of the bottom 12, top 14 and shelf 20 can be modified or processed to enhance certain conditions. For example, one or more biological agents can be applied to one or more portions of the bottom 12, the top 14, and / or the shelf 20. The one or more biological agents include, but are not limited to, an extracellular matrix, or a component thereof, such as laminin, fibronectin, collagen, and any combination thereof. In addition or alternatively, a synthetic agent may be applied. The surface can also be pretreated by, for example, tissue culture treatment or plasma polymerization. As will be appreciated by those skilled in the art, these various processes or modifications may be used in various combinations and utilized depending on the intended purpose.

  The bottom 12, top 14 and shelf 20 are adjacent to form the container 10. In particular, the wall 36 of the top 14 is adjacent to the wall 30 of the shelf 20 positioned adjacent to the top 14. All the shelves 20 are located between the bottom 12 and the top 14. A plurality of adjacent shelves 20 are arranged in a stacked manner, and the walls 30 of the stacked lower shelves 20 are stacked and adjacent to the adjacent upper shelves 20. The bottom 12 is adjacent to a shelf 20 adjacent to the bottom 12, and the shelf 20 must be the lowest shelf 20 stacked when a plurality of shelves 20 are used. The wall 26 of the bottom 12 is adjacent to the adjacent shelf 20. Any known technique such as welding (eg, by ultrasonic welding), gluing, and / or mechanical connection (eg, mating tongues and grooves) may be utilized in any combination to provide a fluid tight seal at these boundaries Can be done. A sealing member, such as a gasket, can be interposed between adjacent components, such as between the top 14 and the adjacent shelf 20. The gasket material can be selected to be gas permeable but liquid impermeable. In addition, one or more exhaust ports 37 may be formed in the bottom 12, the top 14, and / or one or more shelves 20. A gas permeable / liquid impermeable membrane (membrane) may be provided to extend across one or more exhaust ports 37.

  The bottom 12, top 14 and shelf 20 can be sized with various shapes to provide different surface areas for cell culture. Thus, the container 10 can be formed in different sizes and shapes, such as the illustrated flask shape. Alternatively, the container 10 may have a rectangular shape or may be formed in other polygonal or other shapes. Preferably, the container 10 includes a first end 38 configured to support the container 10 in an upright position where the shelf 20 is vertically aligned. This is the preferred state for filling the cell medium to achieve equilibrium. More preferably, the first end 38 is disposed at a position on the container 10 opposite the opening 18 of the tubular neck 16. The first end 38 may be a flat surface or a locus of points that collectively define a seating surface.

  The bottom part 12, the top part 14 and the shelf part 20 form a container 10. The walls 26, 36, 30 of the bottom 12, top 14, and shelf 20, respectively, and the bases 24, 34 of the bottom 12 and top 14 each define a portion of the outer surface of the container 10.

  In order to achieve balance, the flow holes 32b are preferably located proximate to the first end 38. In this method, as shown in FIG. 16, with the cell medium C placed in the container 10 and the container 10 resting on the first end 38, the cell medium C passes through the flow hole 32b. In addition, a volume (V1, V2, V3...) Divided in an equilibrium state between the bottom portion 12, the shelf portion 20, and the top portion 14 is achieved. From equilibrium, the container 10 is placed horizontally resting on the bottom 12, as shown in FIG. The divided amount of cell medium C is eventually spread and distributed to the supporting underlayer defined by the bottom 12 or shelf 20.

  Preferably, the cell medium C is equilibrated in an equal volume between the bottom 12, the ledge 20 and the top 14. An equal spacing, such as the spacing z between the bases 24, 34, 28 of the bottom 12, top 14 and shelf 20, respectively, between each layer, particularly at a location adjacent to the first end 38 of the container 10. Capture an equal amount.

  As shown in FIGS. 13-14, the base 34 of the top portion 14 is formed generally flat. To allow good pipette access, the base 34 of the top 14 has a portion spaced from the base 28 of the adjacent shelf 20, as shown in FIG. The distance z is larger than the distance z seen between the bases 28 or the distance z seen between the base 24 of the bottom 12 and the base 28 of the adjacent shelf 20. As shown in FIG. 17, this increased spacing (spacing q) improves pipette access that contacts the first end 38 throughout the length of the container 10. Thus, using pipette access, the container 10 is rested on the first end 38 to effect post cell culturing, efficient collection of cell media and cells. Increasing the spacing between the base 34 of the top 14 and the base 28 of the adjacent shelf 20, however, is between the top 14 and the adjacent shelf 20, and the amount captured between the shelves 20 and the bottom 12. As a result, a larger amount is captured compared to the amount captured between the adjacent shelves 20.

  Preferably, the base 34 of the top 14 is formed with first, second and third portions 42, 44, 46, respectively. These reduce the amount trapped between the base 34 of the top 14 and the base 28 of the adjacent shelf 20 near the first end 38. In particular, referring to FIG. 18, the first portion 42 is located a distance x from the base 28 of the adjacent shelf 20 and the second portion 44 is separated from the base 28 of the adjacent shelf 20 by a distance y. And the distance x is greater than the distance y. The second portion 44 extends from the first end 38 so as to partially surround the volume adjacent to the first end 38. Preferably, the distance y is equal to the interval z set between the adjacent shelf portions 20 and between the bottom portion 12 and the adjacent shelf portion 20. More preferably, the second portion 44 has a predetermined length L extending from the first end 38, and the predetermined length L is greater than or equal to the length necessary to receive the target equilibrium amount. In this method, as shown in FIG. 16, with the cell medium C equilibrated, the second portion 44 extends at or above the height of the cell medium C (volumes V1, V2, V3). So that ... is equilibrated). In equilibrium, the height of the cell medium C is preferably below the third portion 46. The height of the cell medium C above the second portion 44 and in contact with the third portion 46 results in an unequal amount resulting from equilibration. The arrangement of the first, second and third portions 42, 44, 46 provides good pipette access to the first end 38. This includes access to the volume adjacent to the second portion 44 near the first end 38 by a pipette that passes through a higher area adjacent to the first portion 42. This arrangement also allows for equal volumes to be balanced between the various layers. The first and second portions 42, 44 are each formed flat and can be arranged in parallel, but other shapes (eg, arcs) are possible.

  A third portion 46 of the base 34 extends between and connects the first and second portions 42, 44. Preferably, the third portion 46 is formed flat, but may be formed in other shapes such as an arc shape. Advantageously, the third portion 46 defines a surface suitable for the addition of other indicia such as printing or barcodes. With respect to the plane defined by the second portion 44, the third portion 46 has an angle in the range of about 10-90 °, more preferably an angle in the range of about 10-30 °, more preferably about 20 °. Is arranged at an angle of The third portion 46 disposed to be inclined with respect to the second portion 44 allows the cell medium C to be placed on the first end 38 while the container 10 is stationary on the first end 38. Provide a tapered surface to face. Although an angle α of 90 ° is possible, the angle α is preferably less than 90 ° so as to avoid any cellular medium or cells being trapped at the intersection of the first and third portions 42,46. .

  Use of the second portion 44 defines a recess 48 in the container 10 as shown in FIG. The wall 36 at the top 14 partially borders the recess 48. The recess 48 provides a handle function that allows the user to grip and access the exposed portion of the wall 36.

  As shown in FIG. 2, the gas transmission hole 32 a is preferably linear so as to define a gas flow path 40 in the enclosed volume 22. Preferably, the gas flow path 40 extends in the vicinity of the opening 18 defined in the tubular neck 16. The gas flow path 40 extends through the ledge 20 to allow the gas flow to reach various portions of the enclosed volume 22.

  As described above and as shown in FIG. 18, in the cell culture use state, the container 10 is arranged to rest on the bottom 12. In this position, the cell medium C is spread over the surface of the base 24, 28 of the corresponding bottom 12 or shelf 20 due to its liquid properties. With respect to the bottom 12, the wall 26 provides a fluid containment that maintains the cell medium C on the base 24. With respect to the shelf 20, storage walls 50 are disposed along the edge of each hole 32 to provide storage that avoids cell medium C from passing through any of the holes 32 (32 a, 32 b). And function as a dike. Preferably, the height of the wall 30 of the shelf 20 is larger than the height of the storage wall 50. In this way, an open space can be maintained on each layer of cell medium C between shelves 20.

  Although it is preferred that the same amount be received in each layer within the same container 10, the bottom 12 and shelf 20 may be configured to handle different amounts of cell medium C. The base 24 and the wall 26 of the bottom 12 define a volume for receiving the cell medium C on the bottom 12. The base 28, wall 30 and containment wall 50 of each shelf 20 define a volume for receiving the cell medium C thereon. The bottom 12 and the shelf 20 can each be configured to receive an amount of cell medium C in the range of 4-50 milliliters. Since the bottom 12 does not have holes 32 formed in it, the surface area of the bottom 12 is greater than the surface area of each shelf 20. Thus, the height of the cell medium C layer on the bottom 12 has a height slightly smaller than the height of the cell medium C layer on the shelf 20. The interval z is set in consideration of the capacity of the cell medium C in each layer so that a sufficient head space is secured on the layer of the cell medium C and gas can be appropriately distributed.

There is interest in the cell medium C that is wicked into the corresponding holes 32 by the containment wall 50. As shown in FIGS. 11, 15, and 19, the storage wall 50 is preferably bent along an arcuate longitudinal axis. The bent shape acts against capillary attraction and provides an anti-wicking effect for the containment wall 50.
The hole 32 (32a, 32b) is formed in an arc shape, and the storage wall 50 can be arranged along the edge portion. In addition, the holes 32 can have various shapes, as shown in FIGS. In particular, the gas transmission hole 32a is formed to extend along a part of the side edge 52 of the shelf 20 (FIGS. 11 and 15), or along the entire length of the side edge 52 of the shelf 20. It can be formed to extend (FIG. 19).

The storage wall 50 can be provided with other non-suction features. For example, being inclined with respect to the base 28 of each shelf 20 (FIG. 21); given an arcuate cross section (FIG. 23); and / or non-facing away from the corresponding hole 32 Including a smooth surface 53. As shown in FIGS. 20-24, the non-smooth surface 53 can include various surface interruptions or protrusions. For example, protrusions, small depressions, roughened areas, streaks, and the like. The non-suction feature is intended to prevent capillary attraction.
Additionally or alternatively, the containment wall 50 may be made or prepared to have a hydrophobic portion for repelling the cell medium C. For example, the free edge 54 terminating the containment wall 50 may be prepared to be hydrophobic. Other portions of the containment wall 50 may be treated similarly. In order to further enhance this effect, the portion of the base portion 28 of the shelf portion 20 may be made hydrophilic in order to enhance the retention of the cell medium C on the base portion 28 of the shelf portion 20. These various non-suction features can be used in various combinations.

The container 10 can be provided with additional features or modifications. For example, referring to FIGS. 2 and 25, the tubular neck 16 can be provided at different angular orientations relative to the container 10. As shown in FIG. 2, the tubular neck 16 extends along a longitudinal axis disposed at an angle β with respect to the plane defined by the base 28 of the uppermost shelf 20.
The angle β may be in the range of about 0 ° to 90 °. At 0 °, the tubular neck 16 projects from the side of the top 14, as shown in FIG. At 90 °, the tubular neck 16 has a vertical orientation and protrudes from the base 34 of the top 14 as shown in FIG. With this arrangement, pipette access through the gas flow path 40 to the volume located near the base 24 of the bottom 12 is provided for removal of cell media or cells.
The angle β may alternatively be an acute angle.

  The container 10 is preferably formed so that it can be stacked on other containers. In order to achieve such stacking, the top 14 preferably defines an upper seating surface that is parallel to the seating surface defined by the bottom 12. In this method, two or more containers 10 are stacked and the bottom 12 of the container stacked on top is supported by the top 14 of the container 10 stacked on the bottom. A bead 56 may be defined on the outer surface of the bottom 12 to enhance stability. Correspondingly, the wall 36 of the top 14 may be formed to protrude slightly from the base 34. Wall 36 nests beads 56 and provides lateral stability in the stacked state. It should be noted that the wall 36 of the top 14 is configured to primarily support the weight of the container 10 stacked on top, particularly by protruding from the base 34. Thus, the problem of instability due to the presence of the recess 48 is avoided.

  The base 28 of the shelf 20 is preferably formed flat. Alternatively, the base 28 can be formed in undulating, undulating or other shapes to increase the surface area. In any shape, the layer of cell medium C supported by the ledge 20 is preferably arranged in parallel with the container 10 resting on the bottom 12. The base 24 of the bottom 12 can likewise be formed to be wavy, undulated or other shapes formed to have an increased surface area. In any shape, it is preferred that the layer of cell medium C supported by the bottom 12 is parallel to the other layers of cell medium C.

  The container 10 as an assembly can be provided with a corresponding cap 58 that is configured to be attached to the tubular neck 16 so as to seal the opening 18. Any known configuration that allows attachment is available, such as friction fit, interference fit, screw attachment or bayonet attachment. The cap 58 can be formed of a solid so that there is no exhaust. Alternatively, the cap 58 may be provided with one or more exhaust ports 60 including a gas permeable / liquid impermeable membrane 61. Further, the cap 58 can include one or more regulating valves 62 that allow for selective aseptic connection between the enclosed volume 22 and the external fixture. Further, the cap 58 can include one or more fittings 64 that allow direct and continuous communication with one or more supply tubes T (eg, gas supply tubes).

  In use, the container 10 is placed on the first end 38 and a sufficient amount of cell medium C is introduced into the enclosed volume 22 through the opening 18 to the bottom 12 and each shelf 20. Give the target capacity. A pipette is used to introduce the cell medium C, and the pipette is inserted through the opening 18 into the enclosed volume 22 between the top 14 and the adjacent shelf 20. The introduced cell medium C collects in a volume adjacent to the first end 38, equilibrates between the flow holes 32b, and divides the divided amount of cell medium C corresponding to the bottom 12 and each shelf 20. provide. When equilibrated, the container 10 is adjusted to rest on the bottom 12 and the cell medium C is spread and distributed over the bottom 12 and each shelf 20. A cap 58 may be attached to the tubular neck 16 to seal the opening 18. Thereafter, the container 10 can be placed in an incubator. For transport, the container 10 is preferably held vertically and the cell medium C is preferably accumulated in the enclosed volume 22 adjacent to the first end 38. A plurality of containers 10 may be stacked during the culture.

  The container 10 is placed on the first end 38 to extract the cell medium C for exchange or cell collection purposes. A pipette or other extraction device is introduced to extract the cell medium C from a location adjacent to the first end 38. This allows for complete removal. The pipette is introduced into the enclosed volume 22 between the top 14 and the adjacent shelf 20 and accesses the volume adjacent to the first end 38 for extraction. Cell medium C is aspirated for extraction therefrom, under negative pressure, through flow holes 32b, to a volume adjacent to top 14. Alternatively, cell medium C can be injected through opening 18. Trypsin or other dissociating agents can be utilized to release the cells for harvesting.

10 container 12 bottom 14 top 16 tubular neck 18 opening 20 shelf 22 enclosed volume 24 base 26 wall 28 base 30 wall 32 hole 34 base 36 wall

Claims (36)

A container for culturing cells,
A bottom including a base, the base comprising an upwardly extending wall that at least partially defines a boundary of the base of the bottom;
A top including a base, the top including a downwardly extending wall that at least partially defines a boundary of the base of the top;
A tubular neck defining an opening;
One or more shelves, each shelf comprising a base and an upwardly extending wall that at least partially defines a boundary of the base of the shelves; and
With
The upwardly extending wall of the first shelf of the one or more shelves is adjacent to the downwardly extending wall of the top;
The first shelf is located between the bottom and the top;
The base of each shelf has at least one hole;
The bottom, the top and the one or more shelves aggregate to define an enclosed volume for culturing cells, the tubular neck extends from the container, and the enclosed volume is the A container accessible by said opening in the tubular neck.   The at least one shelf includes a second shelf, the second shelf is located between the bottom and the first shelf, and the bottom wall is adjacent to the second shelf. The container according to claim 1.   The container has a first end, a first volume, and a second volume, wherein the first volume is defined between the top and the first shelf; A predetermined length extending from a first end, and the second volume is defined between the bottom and the second shelf, extending a predetermined length from the first end, the first and The container of claim 2, wherein the second volumes are substantially equal.   4. A container according to claim 3, wherein the opening is aligned to allow pipette access to the first volume near the first end of the container.   A flow hole is formed in each shelf, the first volume communicates with the flow hole in the first shelf, and the second volume communicates with the flow hole in the second shelf. The container according to claim 3.   The base of the top has a first portion that is a first distance away from the base of the first shelf, and the base of the top is second from the base of the first shelf. 4. A container according to claim 3, comprising a second portion separated by a distance of two, wherein the first distance is greater than the second distance.   7. A container according to claim 6, wherein the opening is aligned to allow pipette access to the first volume near the first end of the container.   The container of claim 6, wherein the second portion extends a predetermined length from the first end of the container.   The base of the top includes a third portion, the third portion extending between and connecting the first and second portions of the base of the top. Container.   10. A container according to claim 9, wherein the first and second portions of the base at the top are arranged substantially parallel.   The third portion is substantially flat and is disposed at an angle with respect to a plane defined by the second portion of the base, the angle being in the range of about 10-90 °. Item 10. The container according to Item 10.   The container of claim 11, wherein the angle is in the range of about 10-30 degrees.   The container of claim 11, wherein the angle is about 20 °.   The container of claim 6, wherein the tubular neck extends from the top.   The container of claim 1, wherein the top further comprises at least one vent.   The container of claim 15, wherein a gas permeable membrane extends across the exhaust port.   The container of claim 1, wherein the opening of the tubular neck is elliptical or semi-circular at least partially along the length of the tubular neck.   The tubular neck extends along a longitudinal axis, the longitudinal axis is disposed at an angle with respect to a plane defined by the base of the first shelf, and the angle is in the range of about 0-90 °. The container according to claim 1, wherein the container is inside.   The container of claim 18, wherein the angle is about 0 °.   The container of claim 18, wherein the angle is about 90 °.   The container according to claim 18, wherein the angle is an acute angle.   The container according to claim 1, wherein a gas transmission hole is formed in each of the shelves, and the gas transmission holes of the plurality of shelves are straight and define a gas flow path.   23. A container according to claim 22, wherein the gas flow path extends to the vicinity of the opening defined in the tubular neck.   23. A container according to claim 22, wherein a flow hole is formed in each shelf and the flow hole in each shelf is spaced from the gas transmission hole in each shelf.   The container of claim 1, wherein each shelf includes a storage wall extending upward from the base, the storage wall being defined along at least one hole.   26. A container according to claim 25, wherein the storage wall is bent along an arcuate longitudinal axis.   26. A container according to claim 25, wherein the storage wall is arranged to be inclined with respect to the base of each shelf.   26. A container according to claim 25, wherein the containment wall has an arcuate cross section.   26. The container of claim 25, wherein the containment wall includes a non-smooth surface that faces away from each of the holes.   26. A container according to claim 25, wherein the containment wall terminates at a free edge and the containment wall is at least hydrophobic along the free edge.   31. A container according to claim 30, wherein the base of each shelf has a hydrophilic portion spaced from the storage wall. A container formed according to claim 1;
A cap formed to be attached to the tubular neck;
An assembly comprising:   35. The assembly of claim 32, wherein the cap is provided with an exhaust port.   The assembly of claim 32, wherein the cap is not provided with an exhaust port.   33. The assembly of claim 32, wherein the cap includes a valve to allow selective access to the enclosed volume.   35. The assembly of claim 32, wherein the cap includes means for connecting to one or more tubes.

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